The present invention relates to a bio-molecular microchip and a manufacturing process thereof. More particularly, the present invention is directed to a bio-molecular microchip comprising a substrate, and an amine group-containing bio-molecule that is immobilized onto a glycidyl moiety-containing polyacrylamide gel via covalent bonds formed between the glycidyl moiety of the polyacryl amide gel and the amine group of the bio-molecule by epoxy ring-opening reaction; a manufacturing process thereof.
As is known, oligonucleotide hybridization technique wherein oligonucleotide or target DNA fragment is immobilized on a gel or solid surface, is applied in the various fields. Recently, these oligonucleotide hybridization technique has been applied to DNA sequencing method, and various kinds of research relating to them have been made(Barinaga, M. (1991) Science 253:1489; Cantor, C. R., Mirzabekov, A. and Southern, E. (1992) Genomics 13, 1378-1383; Southern, E. M., Maskos, U. and Elder, J. K. (1992) Genomics 13, 1008-1017; Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J., Shai, N., Shen, N., Yang, R. and Fodor, S. P. A. (1995) Biotechniques 19: 442-447). Also, a number of methods for DNA immobilization on a gel or a solid surface have been developed. They are largely divided into two groups. One is to synthesize oligonucleotides directly on a glass surface, the other is to first synthesize oligonucleotides and immobilized this synthesized oligonucleotides on a solid surface or a gel surface.
For example, Southern et al. has disclosed a method for synthesizing oligonucleotides on a glass surface, comprising: a)immobilizing silicon rubber tubing on a glass surface by using of silicon rubber cement, b)overlapping the glass prepared in a) on a glass to be used in synthesizing oligonucleotides, c) injecting coupling solutions through the channel formed by overlapping to synthesize oligonucleotides in the specific site, and d)rotating sequentially silicon rubber tubing to synthesize oligonucleotides in the rest sites which is blocked(Southern, E. M., Maskos, U. and Elder, J. K. (1992) Genomics 13, 1008-1017; Maskos. U. and Southern, E. M. (1992) Nucleic Acids Res. 20, 1675-1678; Maskos, U. and Southern, E. M. (1993) Nucleic Acids Res. 21, 2267-2268; Williams, J. C., Case-Green, S. C., Mir, K. U. and Southern, E. M. (1994) Nucleic Acids Res. 22, 1365-1367).
Generally, oligonuclotides can be synthesized on a glass surface directly by using photosensitive oligonucleotide synthesis which is applied to DNA sequencing. In the above method, a free hydroxyl group is formed onto the surface which induced photosensitive protector from 5xe2x80x2 hydroxyl group by light emitted through shield mask, and then deoxynucleosides which are protected after forming free hydroxyl group are linked on the surface(Pease, A. C., Solas, D., Sullivan, E. J., Cronin, M. T., Holmes, C. P. and Foder, S. P. A. (1994) Proc. Natl. Acad. Sci. USA 91, 5022-5026; Sapolsky, R. J. and Lipshutz, R. J. (1996) Genomics 33, 445-456; Hoheisel, J. D. (1997) Trends in Biotechnology 15, 465-469; Afftmetrix corp.).
The process for immobilizing oligonucleotides on polyacrylamide of which amide moiety is replaced with hydrazide group, has been developed by Russian scientists. In the above process, 3xe2x80x2-methyluridine at 3xe2x80x2-end of oligonucleotides is activated by sodium periodite(NaIO4) to form dialdehyde groups. Microchip for arranging oligonucleotides in the gel(100xc3x97100xc3x9720 xcexcm) through the above method has been disclosed(Yershov, G., Barsky, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., and Mirzabekov, A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4913-4918; Parinov, S., Barsky, V., Yershov, G., Kirillov, E., Timofeev, E., Belgovsky, A. and Mirzabekov, A. (1996) Nucleic Acids Res. 24, 2998-3004). Also, the method for immobilizing the oligonucleotides on the polypropylene film(PP-NH2), which are aminized by phosphoramidite-based synthesizing process has been disclosed(Matson, R. S., Rampal, J., Pentoney, S. L., Jr., Anderson, P. D. and Coassin, P. (1995) Analytical Biochemistry 224, 110-116). Another method for arranging oligonucleotides is to immobilize 3xe2x80x2-amino altered oligonucleotides on a thin film of silicone dioxide(SiO2), which is formed on a surface of the silicone chip, during the nucleic acid hybridization. In this method, the 3xe2x80x2-amino altered oligonucleotide is fixed onto the thin film of silicone dioxide(SiO2) through an epoxy ring-opening reaction of 3xe2x80x2-amino linkage with a epoxysilane monolayer, which is prepared by treating with 3xe2x80x2-glycidoxypropyltrimethoxysilane(Lamture, J. B., Beattie, K. L., Burke, B. E., Eiggers, M. C., Ehrlich, D. J., Fowler, R., Hollis, M. A. Kosicki, B. B., Reich, R. K., Smith, S. R., Varma, R. S. and Hogan, M. E. (1994) Nucleic Acids Res. 22, 2121-2125). Moreover, a method for inducing covalent bonds has been disclosed. In this method, the covalent bonds are induced by spotting oligonucleotides tailed with homopolymers(dTTP), to activate the thymine base of the oligonucleotide by Ultraniolet(UV) irradiation(Saiki, R. K., Walsh, P. S., Levenson, C. H. and Erlich, H. A. (1989) Proc. Natl. Acad. Sci. USA 86, 6230-6234). Further, a method improved from the above method has been disclosed. This method enables to make a more stable bond by forming amide bone between the amino-linker combined oligonucleotide and the carboxyl groups of the nylon membranes, and to increase efficiency of hybridization (Zhang, Y., Coyne, M. Y., Will, S. G., Levenson, C. H. and Kawasaki, E. S. (1991) Nucleic Acids Res. 19, 3929-3922). The hybridization techniques by adding a radioactive labeled or non-radioactive labeled target DNA to the DNA chip which is developed by methods described above, are successfully used to detect RAS point mutation, cystic fibrosis deletion, and other various point mutation detection as well as DNA sequencing (Cantor, C. R., Mirzabekov, A. and Southern, E. (1992) Genomics 13, 1378-1383; Zhang Y., Coyne, M. Y., Will, S. G. Levenson, C. H. and Kawasaki, E. S. (1991) Nucleic Acids Res. 19, 3929-3933; Hacia, J. J., Brody, L. C., Chee, M. S., Fodor, S. P. A. and Collins, F. S. (1996) Nature Genetics 14, 441-447; Shoemaker, D. D., Lashkari, D. A., Morris, D., Mittman, M. and Davis, R. W. (1996) Nature Genetics 14, 450-456; Sosnowski, R. G., Tu, E., Butler, W. F., O""Connel, J. P. and Heller, M. J. (1997) Proc. Natl. Acad. Sci. USA 94, 1119-1123). These methods can be very effective, especially in genetic mutation tests and genetic polymorphism research (Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M. J., Shai, N., Shen, N., Yang, R. and Fodor, S. P. A. (1995) Biotechniques 19, 442-447; Schena, M., Shalon, D., Davis, R. W. and Brown, P. O. (1995) Science 270, 467-470; Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C. Stern, D., Winkler, J., Lock, D. J., Morris, M. S. and Fodor, S. P. A. (1996) Science 274, 610-614; DeRisi, J., Penland, L. and Brown, P. O.; Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A. and Trent, J. M. (1996) Nature Genetics 14, 457-460). Also, it is predicted that the above methods can be used as ideal tools for diagnosis of infectious and genetic/hereditary diseases as well as mutant analysis such as neoplasm, HLA typing and the like in view of their sensitivity, unity and reproducibility. (Saiki. R. K., Walsh, P. S., Levenson, C. H. and Erlich, H. A. (1989) Proc. Natl. Acad. Sci. USA 86, 6230-6234; Zhang, Y., Coyne, M. Y., Will, S. G., Levenson, C. H. and Kawasaki, E. S. (1991) Nucleic Acids Res. 19, 3929-3933).
Methods for an oligonucleotide arrangement on a glass surface through direct photolithographic synthesis or other synthetic methods are very difficult requiring high-level techniques. One of the problems is that products on solid surface is relatively low, and thus requires further future consideration for adequate and appropriate quantitative analysis.
In contrast, a DNA chip by using a gel of the present invention allows direct arrangement of the oligonucleotides that is synthesized in conventional method, onto a surface, with a desired amount of concentration. A DNA fix capacity of polyacrylamide gel is 50 mM, and 1.5 xcexcmol up to 1.5 mmol concentration can be used for oligonucleotide chip arrangement and hybridization procedure (Yershov, G., Barsky, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishes, A., Dubiley, S., and Mirzabekov, A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4913-4918; Parinov, S., Barsky, V., Yershov, G., Kirillov, E., Timofeev, E., Belgovsky, A. and Mirzabekov, A. (1996) Nucleic Acids Res. 24, 2998-3004). It corresponds to 0. 5 to 50 fmol of oligonucleotide per a square 40xc3x9740xc3x9720 xcexcm. It is 100 times higher than the second dimensional fix capacity of a glass surface (Yershov, G., Barsky, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., and Mirzabekov, A.(1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4913-4918). Therefore, The DNA chip produced using the gel of the present invention can be used in various fields and also can be manufactured conveniently.
The present invention is directed to a bio-molecular microchip comprising a substrate, and an amine group-containing bio-molecule, such as nucleotides, polypeptides or chemical compounds, which is immobilized onto a glycidyl moiety-containing polyacrylamide gel via covalent bonds formed between the glycidyl moiety of the polyacryl amide gel and the amine group of the bio-molecule by epoxy ring-opening reaction.
The reaction of an amine group of a bio-molecule such as a nucleotide, a polypeptide or a chemical compound, with a glycidyl group of a polyacrylamide gel of the present invention, forms a stable covalent bond that bind the bio-molecule onto said gel. A bio-molecular microchip of the present invention may be used for detecting DNA, PCR products or oligonucleotides through hybridization of nucleotides immobilized on the polyacrylamide gel, and also be used for determination of the sensitivity and specificity of the detection.
It is therefore the object of the present invention to provide a process for manufacturing a bio-molecular microchip which comprises immobilizing an amine group-containing bio-molecule onto a glycidyl moiety-containing polyacrylamide gel via covalent bonds formed by an epoxy ring-opening reaction between the glycidyl moiety of the polyacrylamide gel and the amine group of the bio-molecule.
Another object of the present invention is to provide bio-molecular microchips comprising a substrate, and an amine group-containing bio-molecule that is immobilized onto a glycidyl moiety-containing polyacrylamide gel via covalent bonds formed between the glycidyl moiety of the polyacryl amide gel and the amine group of the bio-molecule by epoxy ring-opening reaction.
A still further object of the present invention is to provide a method for detecting DNA fragments conveniently and efficiently by means of hybridization using a DNA or RNA probe.